[unreadable] Blocking both CD28 and CD154 (CD40L) costimulatory molecules can induce long-term allograft survival and tolerance in some but NOT all transplant models. Under stringent conditions (e.g. the skin transplant model, pre-sensitized hosts, diabetic NOD etc), however, this strategy often completely fails to prevent transplant rejection. Thus, identification of key mechanisms that support the CD28/CD154 blockade resistant rejection is of central importance in transplantation, as the eventual success of costimulatory blockade protocol in the clinic most likely hinges on the identification of these escaping mechanisms. I have constructed a new model in which both CD28 and CD154, two conventional T cell costimulatory molecules, are genetically knocked out (CD28/CD154 double knock out or DKO mice). These mice have a normal T cell repertoire and can readily reject fully MHC-mismatched skin allograft. The overall goal of this proposal is to use this model as a linchpin to critically examine the mechanisms supporting CD28/CD154 blockade resistant rejection and to identify new therapeutic targets in tolerance induction. I hypothesized that costimulation mediated via some of the recently described novel costimulatory pathways (e.g. OX40/OX40 ligand, ICOS/ICOS ligand, 4-1BB/4-1BB ligand, CD27/CD70) and/or the novel T cell growth factors (e.g. IL-7, IL-15, IL-21 ) may support the activation of a subset of T cells that drive the rejection process in CD28/CD154 DKO recipients. THE SPECIFIC AIMS ARE: 1. To test the hypothesis that a novel T cell subset(s) is responsible for mediating skin allograft rejection in the CD28/CD154 DKO recipients. 2. To test the hypothesis that costimulation mediated via the novel costimulatory molecule ICOS, CD27, CD134 (OX40), and/or CD137 (4-1BB) supports the activation of a subset of T cells in CD28/CD154 DKO mice that mediate the allograft rejection response. 3. To test the hypothesis that, in the absence of both CD28 and CD154 costimulatory molecules, which often render activated T cells to produce high levels of IL-2, activation of a subset of alloreactive T cells involved in CD28/CD154 independent allograft rejection is supported by novel non-T cell-derived T cell growth factors (e.g. IL-7, IL-15, IL-21). [unreadable] [unreadable] [unreadable]